Figure 5. Inhibition of miR-302d reversed p100 attenuation of Cyclin D1 protein expression, anchorage-independent growth, and promoted G0/G1 cell-cycle progression.

(A–D) UMUC3(p100) cells and p100−/− (p100) cells were stably transfected with construct of anti-miR-302d and the miR-302d were determined by quantitative real-time PCR. The symbol (*) indicates a significant inhibition of miR-302d expression as compared with vector transfectant (p < 0.05) (A and B). The cyclin d1 3′UTR luciferase reporter was transiently transfected into indicated cells and luciferase activity of each transfectant was evaluated. The results were presented as relative cyclin D1 3′-UTR activity and the symbol (*) indicates a significant increase in cyclin D1 3′-UTR luciferase activity as compared with that in vector transfectant (p < 0.05) (C and D). (E and F) The cell extracts were subjected to Western Blot and β-Actin was used as a protein loading control. (G) Flow-cytometry analysis of cell cycle was performed as indicated. (H and I) Anchorage-independent growth was determined in soft agar. And the number of colonies was scored and presented as colonies per ten thousand cells. The symbol (*) indicates a significant increases in comparison to the vector transfectant (p < 0.05).